Ontogenesis of main organs and cloning and expression of two immune-related genes in Litopenaeus vannamei.pdf

1、White leg shrimp Litopenaeus vannamei is a prime aquaculture species, and it covers 90％of western hemisphere culture and is also extensively farmed in Asia. The estimated annual production from Asia during 2002 was 31600

2、0 mt. In China, exotic species (L. vannamei) had introduced during 1988 for experimental purposes and by 1998 is being successfully cultured in commercial scale; over 1000 hatcheries are in operation. The 300000 tons and

3、 210000 tons harvested during 2003 in marine and freshwater farming respectively. To give more understanding for the white leg shrimp, we have investigated the genesis of important systems and organ, including digestive

4、system, nervous system and haematopoetic tissue, furthermore dealed with two important immune related genes.
　　Our investigation on ontogenesis of digestive system in Litopenaeus vannamei, revealed that in non-feeding

5、 stages the yolk mass is the indigenous source of food and until the fifth nauplius (N5) stage retained in foregut cavity, absorption is gradual in nauplius (N) stages while disappearance occurs at N6 stage. The anterior

6、 midgut caecum generates at N3 and it reaches maximum size in Z3; thereafter in successive stages it reduces in size and becomes a single structure in post-larval stage (P3-P4), named the anterior diverticulum. The later

7、al midgut caecum first appears at N6 stage and lengthens along both sides of the midgut and then ramifies to form lobes as larval development. Finally, these lobes become a compact structure, the hepatopancreas. The midg

8、ut lumen is quite apparent in N stages and only becomes wider in successive stages; it is without a cuticle lining and there is no difference among all investigated stages. The foregut in N stages is a simple tube and py

9、loric and cardiac chamber occurs in the third mysis (M3) stage. In the cardiac chamber of post-larval stages, folds were noted and number of that increased gradually. The gastric mill is absent in all investigated larval

10、 stages (P1-P10). The pyloric chamber is smaller than the cardiac chamber and possesses a gland filter, which first appears in M3 stage and forms a more complete shape in forthcoming stages. The hindgut gland first appea

11、rs in P2 and in successive stages it shows more protuberance and complexity. The hindgut or rectum is a simple tube. Cells of the digestive system in all feeding stages show the presence of acid mucins and general protei

12、n, while the inner margins of the foregut and connective tissues of the gland filter are positive for collegen.
　　The nervous system plays pivotal role in co-ordination of body activities and coping with the task of c

13、ontrolling the behavioral activities during larval development. In present study, we give a basic description in the early stage morphology of nervous system in L. vannamei by classical histological techniques. Results r

14、evealed that neuropil first emerged in N1 stage and the front part of nervous system started getting structural shape in N3 where the ganglions were coalesced in front part of head. The complete structure of central n

15、ervous system including proto-, deuto-and tritocerebrum was observed in N6 stage. The structural expansion of nervous system in successive stages was continued and protocerebrum differentiated into anterior and poster

16、ior median protocerebrum at Z3 stage. The optic ganglions, sub-esophageal ganglions and pleon ganglions were distinct in zoea stage. Furthermore, we discuss the relativity between the development of olfactory lobes and t

17、he voluntary movement of larva.
　　It is believed that haematopoietic tissue (HPT) is the source of haemocytes production in crustaceans and defensive role of haemocytes is progressing but investigations on structure o

18、f HPT are limited hence this study has been designed in which we investigated the genesis and morphology of haematopoietic tissue (HPT) and types of haemocyte based on its morphology and cytochemical feature using histol

19、ogical and histochemistical methods. The results revealed that HPT in L. vannamei is not visible histologically in mysis and early two post larvae (P1 & P2), and emerges in P3 which locates in epigastric region. The HPT

20、showed a continuous propagation of cells in successive post larval stages. Four types of cells can be identified in the HPT from adult specimen and mitotic activity is visible on occasion in the HPT. The haemocytes has b

21、een differentiated into five types and some of these types are co-related with the HPT cells. The cytochemical studies suggested that type Ⅰand type Ⅱ cells of haemocytes are PAS positive while scanty presence of prophen

22、oloxidase was observed in type Ⅰ cells similarly the type Ⅰ cells are Sudan Black B positive and rest of the cells showed weak activity against the lipid detective stain. Best of knowledge this is the first effort toward

23、s the genesis of HPT and its relation with circulating haemocytes in L. vannamei.
　　The second part of the study focuses two important immune related genes Cyclophilin A and Proliferating cell nuclear antigen (PCNA);

24、the obtained results are as below respectively.
　　Cyclophilins are ubiquitous proteins with an enzymatic activity of peptidyl-prolyl cis-trans isomerase (PPlase), which play important roles in a variety of stress resp

25、onses. In present investigation a Cyclophilin A gene has been cloned from white leg shrimp (Litopenaeus vannamei) by rapid amplification of cDNA (RACE) and anchored PCR method. The full length of the shrimp L. vannamei C

26、ypA cDNA (LvCypA) is 855 bp, containing 495 bp of open reading frame (ORF), encoding 164 amino acids with an estimated molecular mass of 17.6 kDa. The LvCypA has high homology with that of other known organisms and it ha

27、s four β-strands. The real time PCR analysis revealed that its expression is ubiquitous and expression level is higher in muscle, gill, lymphoid organ and hepatopancrease respectively. The tissues challenged with Vibrio

28、angullarium revealed that in hepatopancrease it was the lowest at 6 h and highest in 48 h. The results revealed that the LvCypA might involve in potential role of innate immunity.
　　The proliferating cell nuclear anti

29、gen (PCNA) has important role in nucleic acid metabolism and its immune responses have also been reported. In present investigation the PCNA was cloned from L. vannamei (LvPCNA). The full length cDNA sequence of LvPCNA e

30、ncodes 261 amino acids showing high identity with PCNAs reported in other species. Tissue distribution of the LvPCNA revealed that its expression is obvious in gill and muscle. In order to understand the response of bact

31、erial challenge, mRNA level of LvPCNA in hepatopancrease, was analyzed after challenged by V. anguillarum. The obtained expression profile of LvPCNA in tissue is variable in different time point. The data enhance the exi